Understanding the diverse biochemistry of the chlorite dismutase family: from O2 to heme

了解亚氯酸盐歧化酶家族的多样化生物化学：从 O2 到血红素

• 批准号: 8964883
• 负责人: Jennifer L DuBois
• 金额: $ 30.44万
• 依托单位: MONTANA STATE UNIVERSITY - BOZEMAN
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8964883
• 关键词: Accounting; Address; Aerobic; Air; Ally; Anthrax disease; Antibiotic Resistance; Archaea; Bacteria; Basic Science; Binding; Biochemical; Biochemical Pathway; Biochemistry; Biology; Carbon; Carbon Dioxide; Catalysis; Cell Respiration; Cell physiology; Cells; Chemistry; Complex; Computing Methodologies; Data; Decarboxylation; Development; Disease; Electrons; Environment; Enzymes; Eukaryota; Eukaryotic Cell; Evolution; Family; Genes; Genetic; Goals; Gram-Positive Bacteria; Growth; Health; Heme; Hydrogen; Hydrogen Peroxide; Individual; Infection; Informatics; Isotopes; Kinetics; Life; Link; Listeriosis; Metabolism; Metals; Methods; Mission; Modeling; Molecular Chaperones; Movement; National Institute of General Medical Sciences; Nature; Organism; Pathway interactions; Perchlorates; Peroxides; Phase; Phenotype; Plague; Porphyrins; Positioning Attribute; Propionates; Protein Family; Proteins; Protons; Public Health; Reaction; Recombinants; Research; Respiration; Series; Side; Staphylococcus aureus; Structure; System; Testing; Time; Tuberculosis; Variant; Virulence; Work; abstracting; base; biodefense; chlorite; cofactor; coproporphyrinogen oxidase; enzyme substrate; ferrochelatase; ferryl iron; heme a; heme biosynthesis; innovation; insight; methicillin resistant Staphylococcus aureus; microbial; novel; pathogen; peroxidation; protonation; protoporphyrin IX; public health relevance; tool

 DESCRIPTION (provided by applicant): Heme is essential for aerobic life and cellular respiration. The pathway by which eukaryotic cells make heme has been known for some time. Prokaryotic heme biosynthesis, by contrast, has been harder to describe. Recently, a pathway for heme biosynthesis that fills all the remaining gaps has been proposed for Gram- positive bacteria. This is a group of organisms that includes numerous important pathogens that are threats to public health and biodefense, such as the causative agents of MRSA, TB, anthrax, and plague. The pathway differs from the canonical one in its final three steps, with the greatest departure at its terminus. The last step is a double oxidative decarboxylation catalyzed by enzymes known as HemQs: a novel subtype of chlorite dismutases (Clds). The latter are heme enzymes that detoxify the chlorite end product of perchlorate respiration, converting it to Cl- to O2. The initial phase of this research resulted in a rigorous description of the structure, mechanism, and biology of O2-generating Clds from both perchlorate respirers and non-respiring pathogens. Leveraging the tools, insights, and scientific team assembled via work on Clds, this proposal aims at providing a description of HemQ function at the level of the individual molecule and extending to the cellular context. As preliminary work, a hemQ strain of Staphylococcus aureus has been generated and shown to be a heme auxotroph and small colony variant (SCV): a phenotype associated with intracellular persistence and antibiotic resistance. In tandem, the HemQ enzyme from S. aureus has been shown to oxidatively decarboxylate two of the four propionate side chains of coproheme III, in a reaction that depends strictly H2O2. Focusing on the S. aureus system, Aim 1 is to understand how HemQ binds and activates coproheme toward oxidative decarboxylation, producing structural and energetic models of SaHemQ in complex with its substrate (coproheme III), intermediate (harderoheme) and product (heme b). Aim 2 is to test a mechanism for HemQ's reaction, in which coproheme is both substrate and cofactor in the peroxidation. Time-resolved and kinetic isotope methods will be used to examine a series of hypotheses invoking a ferric-hydroperoxy intermediate and intramolecular hydrogen atom transfer. Finally, aim 3 uses genetic, cell-based, and biochemical methods to understand HemQ's function in the context of the cell and evolution. We expect completion of the proposed work to define the ultimate step of a pathway that is absolutely fundamental to aerobic life, essential for robust pathogenic growth, and clinically connected to the development of persistence and antibiotic resistance.

 描述（由申请人提供）：血红素对于有氧生命和细胞呼吸至关重要。真核细胞制造血红素的途径已知已有一段时间了。相比之下，原核血红素生物合成更难描述。最近，针对革兰氏阳性细菌提出了一种填补所有剩余空白的血红素生物合成途径。这是一组生物体，包括许多对公共健康和生物防御构成威胁的重要病原体，例如耐甲氧西林金黄色葡萄球菌、结核病、炭疽和鼠疫的病原体。该路径与典型路径的不同之处在于最后三个步骤，最大的偏离是在其终点。最后一步是由称为 HemQs 的酶催化的双重氧化脱羧：HemQs 是亚氯酸盐歧化酶 (Clds) 的一种新亚型。后者是血红素酶，可解毒高氯酸盐呼吸的亚氯酸盐终产物，将其转化为 Cl- 至 O2。这项研究的初始阶段对高氯酸盐呼吸器和非呼吸性病原体产生氧气的Clds的结构、机制和生物学进行了严格的描述。利用通过 Clds 工作组建的工具、见解和科学团队，该提案旨在提供个人层面的 HemQ 功能描述 分子并扩展到细胞环境。作为初步工作，金黄色葡萄球菌的 hemQ 菌株已被生成，并被证明是血红素营养缺陷型和小菌落变体 (SCV)：一种与细胞内持久性和抗生素耐药性相关的表型。同时，来自金黄色葡萄球菌的 HemQ 酶已被证明可以使 coproheme III 的四个丙酸侧链中的两个氧化脱羧，该反应严格依赖于 H2O2。重点关注金黄色葡萄球菌系统，目标 1 是了解 HemQ 如何结合并激活 coproheme 进行氧化脱羧，生成 SaHemQ 及其底物 (coproheme III)、中间体 (harderoheme) 和产物 (heme b) 复合物的结构和能量模型。目标 2 是测试 HemQ 反应的机制，其中粪血红素既是过氧化反应的底物又是辅助因子。时间分辨和动力学同位素方法将用于检验一系列涉及铁-氢过氧中间体和分子内氢原子转移的假设。最后，目标 3 使用遗传、细胞和生物化学方法来了解 HemQ 在细胞和进化背景下的功能。我们期望完成拟议的工作，以定义一条途径的最终步骤，该途径对于有氧生活绝对至关重要，对于致病菌的强劲生长至关重要，并且在临床上与持久性和抗生素耐药性的发展相关。